PMID: PMC4261947-1-1

 

    Legend: Gene, Sites

Title : Qualitative Analyses ofGlycopeptides from PSA and PSA-HighpI Isoform

Abstract :
  1. Prostate specific antigen in the samples analyzedhere was identified with sequence coverages of 85.6 and 86.5% from PSA and PSAH samples, respectively (data not shown)
  2. Peptides containingthe glycosylation sequon (N69KS) were not detected in theproteomics analysis, thus suggesting that the N69 residueis glycosylated
  3. Protein identifications in proteomics analysis werebased on an ion score cutoff higher than 30 and a minimum number ofpeptides of two
  4. According to spectral count data generated by Scaffoldsoftware, spectral counts of PSA accounted for 86% of the total spectralcount data (15 identified peptides ), whereas the spectral counts ofPSAH accounted for 78% of the total spectral count data (17 peptides )
  5. Other proteins that were detected in the case of the PSA sample were triosephosphate isomerase (6% of total spectral counts, three peptides ), prostaglandin-H2 d-isomerase (3% of the total spectral countdata, two peptides ), and growth/differentiation factor 15 (5% of totalspectral count data, two peptides )
  6. Other proteins detected in thecase of the PSAH sample were triosephosphate isomerase (12% of thetotal spectral count data, eight peptides ), prosaposin (5% of thetotal spectral count data, two peptides ), prostaglandin-H2 d-isomerase (2% of the total spectral count data, two peptides ), andgrowth/differentiation factor 15 (3% of the total spectral count data,two peptides )
  7. Furthermore, GlypID analysis using these proteins didnot identify any valid glycopeptides associated with above-mentioned proteins , yet they all have the glycosylation sequences
  8. Therefore,all glycopeptides reported here are unique to PSA and are not originatingfrom the other proteins observed in the LC–MS/MS analysis
  9. Additionally, the Y1 ions reported here are unique to PSA and arenot from any contaminant proteins observed in the LC–MS/MSanalysis
  10. Since PSA is known to have only one N-linked glycosylationsite at Asn69 , it is adequate to interpret its glycosylations withCID or HCD tandem MS. If the CID or HCD spectra include glycan fragmentationor glycopeptide diagnostic ions in addition to the expected Y1 ion( peptide + GlcNAc) , then the glycopeptides associated with Asn69 wereconfirmed
  11. For example, a Y1 ion associated with Asn69 detected atan m/z value of 602 or 601 correspondedto doubly charged NKSVILLGR + GlcNAc
  12. Hexose rearrangement could occurduring CID tandem analyses
  13. It is observed in lowabundance (<8%) when Y1 ion intensities are incredibly high
  14. However,hexose rearrangement is not the case in this study because Y1 ionintensities associated with the PSA glycosylation site are not thathigh, as can be seen in CID MS/MS scans (SupportingInformation Figure 1)
  15. In cases when the theoretical m/z values of glycopeptides were used,they were confirmed by matchingthe retention times of the glycopeptides that were confirmed by tandemMS
  16. Using a 5 ppm cutoff mass accuracy allowed glycopeptides thathave similar molecular weights to be distinguished
  17. For example, aglycopeptide possessing 2 Fuc residues is only 1 Da different fromthat possessing an NeuAc residue , which can be differentiated usingthis 5 ppm cutoff
  18. Moreover, their retention time differences werealso used to distinguish them, since a glycopeptide containing NeuAcis retained longer by LC than a counterpart glycopeptide containing2 Fuc
  19. Overall, the use of the aforementioned criteria results inan overall averaged mass accuracy of 3.05 ppm for all identified glycopeptides
  20. In total, tryptic digestion of the samples resulted in the formationof three peptide backbones containing the glycosylation site , namely,NKSVILLGR (17–23 min), AVCGGVLVHPQWVLTAAHCIRNK(26–21 min), and AVCGGVLVHPQWVLTAAHCIRNKSVILLGR(31–36 min), as shown in Figure 1A
  21. There are 56 glycopeptides observed for PSA , as summarized in Table 1
  22. Because 55 of the structures were identified onthe NKSVILLGR backbone, the discussion will be mainly focused on thispeptide backbone
  23. One missing glycoform associated with the NKSVILLGRbackbone is HexNAc3Hex3dHex1NeuAc1 , which was detected on theAVCGGVLVHPQWVLTAAHCIRNK backbone
  24. Figure 1B–D illustrates averaged full MS with observedN-glycans detected on the NKSVILLGR (=P) backbone
  25. There were 11 neutralN-glycans observed at 17.5 min (Figure 1B),36 monosialylated and 2 sulfated/phosphorylated N-glycans were observedat 19.5 min (Figure 1C), and 6 disialylatedN-glycans were detected at 22 min (Figure 1D)
  26. The m/z values (charge state= 3) of these glycopeptides were listed with confirmed glycan structuresif they were supported by tandem MS data
  27. Otherwise, the most likelystructures were described as previously reported
  28. There were 32 N-glycansconfirmed by tandem MS data, as shown in Table 1
  29. For PSAH, similar retention times were detected withdifferenttypes of N-glycans, as mentioned above (Figure 2A)
  30. In total, 57 N-glycans were identified from three peptide backbones,as summarized in Table 2
  31. All of the N-glycanswere detected on the NKSVILLGR backbone except HexNAc6Hex4dHex2NeuAc1 ,which was detected only on the AVCGGVLVHPQWVLTAAHCIRNKbackbone
  32. Figure 2B–D depicts an averagedfull MS with identified N-glycans associated with the NKSVILLGR backbone
  33. There were 15 neutral N-glycans observed at 17.5 min (Figure 2B), 29 monosialylated and 2 sulfated/phosphorylatedN-glycans observed at 19.5 min (Figure 2C),and 10 disialylated N-glycans detected at 22 min (Figure 2D)
  34. Manual annotations of the CID MS/MS of 37 glycopeptidesare depicted in Supporting Information Figure1
  35. Assigning glycan structure from CID MS/MS is achievablebecausethe mass difference between the glycan residues of glycopeptides ,such as Hex, HexNAc, Fuc, or NeuAc, is rather large
  36. Also, sequencingN-glycan structures follows some rules because specific glycosyltransferases/exoglycosidasesare involved in trimming or attachment of each glycan residue duringthe biosynthesis of N-glycans
  37. The precursor m/z values of glycopeptides were confirmed using a 5 ppm cutoffmass accuracy because the detection of precursor ions was achievedby an Orbitrap FTMS analyzer
  38. The intriguing features of N-glycansassociated with PSA are thepresence of an GalNAc residue instead of Gal followed by GlcNAc andsulfation/phosphorylation
  39. In this study, many N-glycanswith these characteristics were detected, some of which were confirmedby tandem MS, as depicted in Figure 3
  40. Theglycan fragments of a glycopeptide with m/z 1034.1406 were assigned with diagnostic ions originatingfrom glycan residues (Figure 3A)
  41. The annotationof this tandem MS demonstrates that this glycopeptide is core fucosylatedand monosialylated followed by either a Gal or GlcNAc residue
  42. Thecore fucosylation was confirmed on the basis of the existence of aY1+2 ion (peptide backbone + GlcNAc) with fucose, whichhas an m/z value of 675.3
  43. One ofthe fragment peaks with an m/z valueof 1303.95 represents the loss of a GalNAc residue
  44. Also, we confirmedthat there are two co-eluting isomeric glycopeptides
  45. In the low m/z region , diagnostic ions with m/z values of 495.16 and 698.35 confirmedGalNAc + NeuAc and GlcNAc + GalNAc + NeuAc glycan structures, respectively
  46. In addition, the presence of a 657.19 m/z value affirms the presence of a GlcNAc \+ Gal \+ NeuAc lycan structure
  47. Therefore, the sialylation followed by Gal and GalNAc residues co-eluted,as confirmed in this single tandem MS. Also, on the basis of the assignmentof fragment ions of glycans, we distinguished between the attachmentof GalNAc followed by antenna GlcNAc and bisecting structures
  48. Whena series of HexNAc fragmentation were detected as associated withantenna GlcNAc without assignment of a Glc residue , the presence ofGalNAc on an antenna GlcNAc was confirmed
  49. Regarding bisection structures,the presence of 2GlcNAc + Man + GlcNAc, 2GlcNAc + 2Man + GlcNAc, and2GlcNAc + 3Man + GlcNAc was detected, which correspond to m/z values of 886, 967, and 1048, respectively
  50. Figure 3B illustrates annotated tandemMSof a sulfated/phosphorylated glycopeptide corresponding to m/z 963.7589
  51. The sulfation/phosphorylationon a GlcNAc residue was determined on the basis of the detection of fragment ions at m/z 1303.87 and1344.45, the difference of which corresponds to a doubly charged sulfateor phosphate ion
  52. Because the peak at m/z 1344.45 originated from the loss of a GalNAc residue , this suggeststhat the occurrence of sulfation/phosphorylation is on a GlcNAc residue
  53. Also, the presence of m/z valuesof 1221.05 and 1262.04 supports the occurrence of sulfation/phosphorylationon a GlcNAc residue
  54. The difference between these two assignmentsof sulfation/phosphorylation was 0.5 Da, which fits well within themass accuracy of CID MS/MS
  55. On the other hand, no diagnostic ionsrepresenting a sulfated/phosphorylated glycan residue were observedfrom HCD MS/MS, as shown in Supporting InformationFigure 2
  56. Overall, more of core-fucosylated and/or disialylatedglycoforms were identified in PSAH than in normal PSA
  57. Moreover, highlybranched glycan structures such as tri- or tetra-antennary structureswere more abundant in PSAH than in normal PSA
  58. These observationsare in agreement with previously published results
  59. Glycoform differences between PSA and PSAH are illustrated in Supporting Information Figure 3
  60. Therewas a difference in the number of identified N-glycans fromthe three peptide backbones, as shown in Tables 1 and 2
  61. This might be due to the fact thatthe ionization efficiency of peptides becomes substantially higheras the mobile phase’s organic content increases
  62. As a result,some of the glycopeptides with minor intensities would not be subjectedto tandem MS experiments
  63. This observation is supported by the datashown in Supporting Information Figure 4
  64. Quantitative results of all identified glycopeptides associatedwith the three peptide backbones are illustrated in Supporting Information Figure 4A,B for PSA and PSAH, respectively
  65. Glycopeptides with relatively low intensities associated with theNKSVILLGR backbone were not detected in the other two peptides backbones,which is believed to be mainly due to competitive ionization
Output (sent_index, trigger, protein, sugar, site):
  • 10. glycopeptide, , -, -, glycopeptide
  • 10. glycopeptides, , -, -, glycopeptides
  • 10. ion, , -, -, peptide
  • 14. glycosylation, , -, -, site
  • 15. glycopeptides, , -, -, glycopeptides
  • 16. glycopeptides, , -, -, glycopeptides
  • 17. aglycopeptide, , -, -, aglycopeptide
  • 18. glycopeptide, , -, -, glycopeptide
  • 18. glycopeptide, , -, a counterpart glycopeptide containing2 Fuc, glycopeptide
  • 19. glycopeptides, , -, -, glycopeptides
  • 2. Peptides, , -, -, sequon
  • 2. glycosylation, , -, -, sequon
  • 20. glycosylation, , -, -, site
  • 21. glycopeptides, , -, -, glycopeptides
  • 26. glycopeptides, , -, -, glycopeptides
  • 35. glycopeptides, , -, -, glycopeptides
  • 37. glycopeptides, , -, -, glycopeptides
  • 40. glycopeptide, , -, -, glycopeptide
  • 41. glycopeptide, , -, -, glycopeptide
  • 44. glycopeptides, , -, -, glycopeptides
  • 48. fragmentation, , fragmentation, associated withantenna GlcNAc, -
  • 50. glycopeptide, , -, -, glycopeptide
  • 62. glycopeptides, , -, -, glycopeptides
  • 64. glycopeptides, , -, -, glycopeptides
  • 7. glycopeptides, , -, -, glycopeptides
  • 8. glycopeptides, , -, -, glycopeptides
Output(Part-Of) (sent_index, protein, site):
  • 14. PSA, site
  • 40. Theglycan, fragments
*Output_Site_Fusion* (sent_index, protein, sugar, site):

 

 

Protein NCBI ID SENTENCE INDEX